The present study was aimed at assessing the validity and the reliability of the Sinhala version of the Utrecht Work Engagement Scale-Student Version (UWES-S) among collegiate cycle students in Sri Lanka.The 17-item UWES-S was translated to Sinhala and the judgmental validity was assessed by a multi-disciplinary panel of experts. Construct validity of the UWES-S was appraised by using multi-trait scaling analysis and exploratory factor analysis (EFA) on data obtained from a sample of 194 grade thirteen students in the Kurunegala district, Sri Lanka. Reliability of the UWES-S was assessed by using internal consistency and test–retest reliability. Except for item 13, all other items showed good psychometric properties in judgemental validity, item-convergent validity and item-discriminant validity. EFA using principal component analysis with Oblimin rotation, suggested a three-factor solution  explaining 65.4% of the total variance for the 16-item UWES-S (with item 13 deleted). All three subscales show high internal consistency with Cronbach’s α coefficient values of 0.867, 0.819, and 0.903 and test–retest reliability was high (p < 0.001). Hence, the Sinhala version of the 16-item UWES-S is a valid and a reliable instrument to assess work engagement among collegiate cycle students in Sri Lanka. Keeping on par with the emerging trend towards a positive psychology focusing on optimal functioning rather than on malfunctioning, a growing enthusiasm is evident in student engagement research during the last few decades .Work engagement is defined as, “a positive, fulfilling, work-related state of mind that is characterised by vigor (VI), dedication (DE), and absorption (AB). Rather than a momentary and specific state, engagement refers to a more persistent and pervasive affective-cognitive state that is not focused on any particular object, event, individual or behaviour” .Engagement of students in their academic environment has been studied in numerous disciplines –10 and tAmongst the different assessment tools of student work engagement, the most commonly used instrument is the Utrecht Work Engagement Scale-Student Version (UWES-S) , 18–22, Even though a plethora of research has been conducted on work engagement among varying student populations across the globe, there is a paucity of literature in the South Asian context with no published literature on work engagement among Sri Lankan students, mainly owing to the lack of validated assessment tools. In the context of ever-increasing burden of mental health problems in the Sri Lankan collegiate cycle students –26, explThe 17-item UWES-S is a self-administered questionnaire (SAQ) with six, five, and six items assessing VI, DE and AB subscales respectively, measured on a seven-point Likert scale anchored by the response options from 0 (never) to 6 (every day).The 17-item UWES-S was translated to Sinhala by using the forward–backward translation method –29, invoThe synthesised forward translation of UWES-S was pre-tested among a sample of 25 grade thirteen students outside the study setting. In a subsequent structured interview, the clarity in understanding, acceptability, and comprehension of items and feasibility of using the questionnaire were assessed. None of the items of the questionnaire was claimed to be difficult to understand.The face, content, and consensual validity were assessed. Using a modified Delphi technique, a multi-disciplinary panel of experts in the fields of psychiatry, psychology, public health, teaching, student counseling, and medical education has assessed each item on its relevance, appropriateness, and acceptability in the local context for assessing burnout among collegiate cycle students based on a rating scale from 0 (strong disagreement) to 10 (strong agreement). At the end of this iterative process, except for the item 13 stating, “When studying, I am very resilient, mentally”, all other items had a median score more than 7 for all the aspects. Thus, it was decided to include all 17 items for the assessment of construct validity.This school-based, cross-sectional validation study was conducted in the Kurunegala district, North Western province, Sri Lanka from May 2014 to April 2015. In Sri Lanka, the collegiate cycle in the education system consists of grade twelve and thirteen. Three Sinhala medium government schools in the Kurunegala district having all four collegiate cycle subject streams, viz., Science, Arts, Commerce and Technology were selected.From the selected schools, three grade thirteen classes each were selected representing both male and female students studying in all four subject streams. A total of 194 students participated in the study in their classrooms and each participant filled the SAQ independently. The response rate was 100.0% and 55.2% (n = 107) of the sample were females. The mean age was 18.3 years (SD = 0.43 years). The majority of students were studying in the Science stream . The numbers of students in the Arts, Commerce and the Technology streams were 60 (30.9%), 41 (21.2%), and 15 (7.7%) respectively.Data were analysed by using the Statistical Package for Social Sciences (SPSS) version 17.0. Preparatory data analysis showed that there were no violations of the assumptions related to the data analytic techniques. Given that the participants to variables ratio was 11.4, the sample size was adequate for factor analysis . Even thItem-scale correlations were analysed and item-convergent and item-discriminant validity were assessed. A stringent criterion of correlation of 0.40 or greater between an item and its own subscale was considered as a success for assessing item-convergent validity. Furthermore, in assessing item-discriminant validity, items that correlated significantly higher (more than 1.96 standard errors) with its own subscale than with the other two subscales, were considered as scaling successes.EFA was conducted by Principal Component Analysis (PCA) with Oblimin rotation. Kaiser’s criterion/eigenvalue, scree plot and parallel analysis were used to decide the number of factors to retain. The factors that lead to a meaningful interpretation and theoretical sense were ultimately selected.Reliability was assessed using internal consistency and test–retest reliability by re-administering the SAQ to a sub sample of 22 grade thirteen students after 2 weeks.Descriptive statistics of UWES-S subscales are given in Table Table Item 13 was found to have poor psychometric properties in judgemental validity, item-convergent validity, and item-discriminant validity. Deletion of item 13 from the subscale also improved the internal consistency. Hence, both 17-item UWES-S and 16 items of the UWES-S (item 13 deleted) were subjected to PCA. The Kaiser–Meyer–Olkin Measure were 0.851 and 0.855 respectively and Bartlett’s test of sphericity reached statistical significance (p < 0.001) for both versions.Only three factors had eigenvalues more than 1.0, which cumulatively explained 63.9% and 65.5% of the total variance for the 17-item and 16-item UWES-S versions respectively. For both versions, the parallel analysis showed only two factors with eigenvalues exceeding the corresponding criterion values and the screeplot showed a clear break after the second factor.However, for both versions, the component matrix in PCA with unrotated loadings revealed a number of items of UWES-S loading on the third factor with values greater than 0.3. Furthermore, the pattern matrix generated in PCA using direct Oblimin rotation revealed that seven items had loading values more than 0.3. Based on this collective evidence, it was decided to ‘force’ a three-factor solution for further investigation.Even though both Varimax rotation and Oblimin rotation was used, it was decided to report statistics related to Oblimin rotation as the factors were strongly correlated > 0.3). The rotated three-factor solution revealed a simple structure with all three factors showing a number of strong loadings. In the 17-item UWES-S version, the three-factor solution explained a total of 63.9% of the variance, with factor one contributing 41.4%, factor two contributing 14.9%, and factor three contributing 7.6%, whereas, the corresponding values for the 16-item UWES-S version were 65.4, 44.0, 14.2, and 7.2%. Table . The rotThe interpretation of three-factor solution of the UWES-S with item 13 deleted, was consistent with previous research on the UWES-S, with subscale items of VI, DE, and AB loading strongly on three different factors. Hence, the three-factor solution with item 13 deleted was considered as the validated Sinhala version of the UWES-S.The impact of each item on the related subscale was assessed by computing Cronbach’s α when the respective item is deleted. None of the items in DE subscale and AB subscale improved the overall Cronbach’s α value; however, deletion of item 13 improved the Cronbach’s α value from 0.696 to 0.867. Hence, item 13 was removed from the UWES-S and the reliability was re-assessed for internal consistency and all three subscales show high internal consistency with Cronbach’s α coefficient values of 0.867, 0.819, and 0.903 for VI, DE, and AB subscales respectively.There were strong, positive statistically significant (p < 0.001) correlations for each of the three subscales of the UWES-S in test–retest reliability assessment. The correlation coefficients were 0.831, 0.866 and 0.839 for the VI, DE, and AB subscales respectively.The present study was designed to validate the Sinhala version of the UWES-S among Sri Lankan collegiate cycle students addressing an important research vacuum on work engagement research in high school students in the South Asian context. A cross-sectional study design and the sample size of the study deemed appropriate with the study objective.In assessing the judgemental validity, item 13  had the least median rating score and the main concern raised regarding the item was the difficulty in explaining the phrase “mentally resilience” in Sinhala language. The global literature suggests that modified versions of the UWES-S with removal of several items have been used in work engagement among high school, college, and university students , 22, 31.Even though the three-factor structure of the Utrecht Work Engagement Scale (UWES) has been confirmed in a large number of studies –36, the The three-factor solution of the original 17-item UWES-S, explained 63.9% of the variance, whereas the three-factor solution of the UWES-S with item 13 deleted, explained 65.4% of the variance. Furthermore, the interpretation of the three-factor solution of the UWES-S with item 13 deleted, was consistent with previous research on the UWES-S with subscale items of VI, DE, and AB loading strongly on three different factors , 22.In assessing the internal consistency, deletion of item 13 improved the Cronbach’s α value from 0.696 to 0.867 and all three subscales showed high internal consistency. This finding is consistent with findings of studies using the Portuguese version , SpanishThe present study also revealed that there were statistically significant strong positive correlations for each of the three subscales of UWES-S in the test–retest reliability assessment. Though these findings are consistent with the previous study findings , the valIn conclusion, the study findings indicate that the Sinhala version of the 16-item UWES-S is a valid and a reliable instrument to assess work engagement among collegiate cycle grade thirteen students in Sri Lanka. Due to its brevity, ease of administration and sound psychometric properties, it could be used as an effective screening tool at the school level. The study findings pave the way to establish the initial evidence base for the relevance and the applicability of the concept of work engagement in the South Asian context.This study has some limitations. Given that the study sample was recruited not using a probability sampling technique in a selected district of Sri Lanka, the generalisability of the study findings to other populations should be done with caution, considering the variations in educational and cultural contexts. Furthermore, given that the three-factor structure of the UWES-S is established in the present study, confirmatory factor analysis procedures are recommended for future studies.


However, such cement is relatively weak, restricting its use to areas of low mechanical stress. Wollastonite fibers (WF) have been used to improve the mechanical strength of biomaterials. However, the biological properties of WF remain poorly understood. Here, we tested the response of osteoblast-like cells to being cultured on CPC reinforced with 5% of WF (CPC-WF). We found that both types of cement studied achieved an ion balance for calcium and phosphate after 3 days of immersion in culture medium and this allowed subsequent long-term cell culture. CPC-WF increased cell viability and stimulated cell differentiation, compared to nonreinforced CPC. We hypothesize that late silicon release by CPC-WF induces increased cell proliferation and differentiation. Based on our findings, we propose that CPC-WF is a promising material for bone tissue engineering applications.Calcium phosphate cement (CPC) that is based on  Recent rs [WF]) , 12. Alsrs [WF]) .α-TCP-based CPC can be enhanced by the addition of WF (CaSiO3) [ wollastonite hydrolysis. Motisuke et al. [ Wollastonite exhibits excellent in vitro bioactivity [The mechanical properties of (CaSiO3) . This rae et al.  found thactivity , as demoactivity . in vitro. The study evaluated the potential of the two types of cement as a substrate for the differentiation, adhesion, and proliferation of osteoblast-like cells.The purpose of the present study was to compare the cytocompatibility of CPC and CPC-WFα-TCP was obtained by solid-state reaction as published elsewhere [3) and Mg-free monetite (CaHPO4) was calcined at 1300°C and milled to achieve a granulometric distribution of 1.33 to 10 μm and a mean particle size of 4.93 μm. WF were synthesized by the salt fusion method [3, Sigma Aldrich) and electronic grade silicon dioxide  were intimately mixed with a NaCl/KCl flux and calcined at 950°C and salt was washed out with deionized water. To prepare CPC-WF, α-TCP and WF were mixed at a α-TCP to WF ratio of 5% (w/w). This 5% WF ratio was assumed optimal for increasing the mechanical properties of CPC, as shown in our previous work [lsewhere . Brieflyn method . In summous work . CPC comous work .2 atmosphere. The medium was changed every 2-3 days and cells were used for the experiments until the fourth passage.Osteoblast-like cells were obtained from the calvaria of 20-day-old Lewis rats by the explant method. This experimental protocol was approved by the Ethics Committee on Animal Use of UNICAMP (CEUA 2606-01). Before each cell isolation procedure, animals were sacrificed, and the calvaria is removed and immersed in DMEM  supplemented with antibiotics . The soft tissue was removed with a scalpel. Next, the calvaria was fragmented and immersed in flasks containing DMEM supplemented with antibiotics and 10% fetal bovine serum . The culture flasks were kept at 37°C in a 5% CO4 cells per disk. As negative controls, cells were cultured in a polystyrene 24-well plate. After incubation, viable cells were evaluated using a colorimetric MTT -2,5-diphenyl tetrazolium bromide)  reduction assay for mitochondrial activity . After 4 h of incubation in the presence of MTT, absorbance readings were made using a microplate reader  at a wavelength of 570 nm.Cell viability was evaluated after 1, 7, and 14 days in culture. After the ion balance period (described above), cells were seeded on the materials at a concentration of 3 × 10Cell morphology on CPC disks was analyzed by scanning electron microscopy (JEOL JXA-840A). For this purpose, samples were fixed in a solution of 4% paraformaldehyde , 2.5% glutaraldehyde , 0.03% picric acid , and 1% tannic acid  in DMEM for 30 min. Next, samples were washed in Phosphate Buffer Saline (PBS), postfixed in 1% osmium tetroxide , and dehydrated in an increasing ethanol series. Samples were then critical-point dried  and sputtered with gold .β-glycerol phosphate, 0.1 mM ascorbic acid, and 1 nM dexamethasone. ALP activity was measured at 7, 10, and 14 days after incubation. Before the assay, disks were washed twice with PBS and cells were lysed by sonication and Triton X-100, according to the manufacturer's instructions . Production of p-nitrophenol was measured by absorbance in a microplate reader  at a wavelength of 405 nm.Alkaline phosphatase (ALP) activity was assayed to identify the early stage of osteoblast differentiation in the various materials. ALP activity can indicate whether cells are osteoblast-like cells. The colorimetric method used was based on the conversion of p-nitrophenyl phosphate into p-nitrophenol in the presence of ALP. Cells were seeded on disks with DMEM supplemented with 3 mM 2+, Si, and P ion levels, aliquots of medium were removed during the ion balance period (during days 1 and 3 of culture) and during cell culture  and analyzed by ICP-OES .To determine Cap < 0.05) were identified, Tukey's post hoc test was applied . All experiments were performed in quintuplicate.All data were compared by one-way analysis of variance (ANOVA). Whenever statistically significant differences (p < 0.01) was observed on CPC-WF than on either polystyrene plates (negative control) or CPC   .2+ and release of P ions after 1 day of immersion in DMEM. On day 3 of the ion balance period, we detected an increase in Ca2+ concentration and reduction in the release of P ions. ICP-OES data indicated that the Ca2+ and P concentrations in CPC and CPC-WF samples were similar to those of the negative control after 3 days of ion balance in DMEM. There was a steady decrease in the rate of Si release from CPC-WF samples throughout the immersion period  were at similar concentrations after 7 and 14 days of culture. These data suggest that ion levels become balanced by day 7 of culture (During the ion balance period, we noted a depletion of Can period . All of  culture . For thiIn vitro analysis of biomaterials in cell culture is a valuable tool for understanding how recently developed materials elicit adverse reactions at the cellular level [ar level , 16. Suiar level . Here, wα-TCP-based cement improves bioactivity by hastening the formation of a dense apatite layer over the cement surface. Based on our data, together with the work of Morejón-Alonso et al. [We found that both CPC and CPC-WF enable cell adhesion, spreading, and increased viability over time. However, after 14 days in culture, cell viability was significantly higher on CPC-WF than CPC. Cell adhesion and proliferation depend on both the physical and chemical characteristics of their substrate . CPC undo et al. , we propIn addition to the physical characteristics of CPC, which favor cell adhesion and proliferation, the chemical composition of cement also influences cellular responses. Mestres et al.  studied The ability to induce differentiation of osteoblast-like cells is fundamental to the success of biomaterials for bone tissue engineering. ALP is known to be an early marker of osteoblast differentiation . This enTaken together, our data suggest that CPC samples are likely to achieve ion balance with their culture medium over time. Only after this balance has been achieved, can cells be cultured and maintained for long periods. All types of CPC tested here were biocompatible for osteoblast-like cells. The addition of WF increased cell viability, as well as the activity of ALP, which could be directly related to silicon release in the medium. Our findings suggest that CPC-WF could be applied as a scaffold in bone tissue engineering strategies.


A CVD-grown boron-doped diamond film was polished to a surface roughness of  Chemical vapour deposition (CVD) grown boron-doped nanocrystalline diamond (B-NCD) is an attractive material for the fabrication of high frequency superconducting nanoelectromechanical systems (NEMS) due to its high Young’s modulus. The as-grown films have a surface roughness that increases with film thickness due to the columnar growth mechanism. To reduce intrinsic losses in B-NCD NEMS it is crucial to correct for this surface roughness by polishing. In this paper, in contrast to conventional polishing, it is demonstrated that the root-mean-square (RMS) roughness of a 520 nm thick B-NCD film can be reduced by chemical mechanical polishing (CMP) from 44.0 nm to 1.5 nm in 14 hours without damaging the sample or introducing significant changes to the superconducting transition temperature,  Diamond is a material of extreme properties of particular use in the fabrication of nano-electro-mechanical systems (NEMS) for the study of macroscopic quantum objects . The thel growth –5. The ul growth –8 or usel growth ,10.To detect the displacement of a doubly clamped NEMS device, it may be directly incorporated into the loop of a superconducting quantum interference device (SQUID) as successfully demonstrated by Etaki et al. . This inIf B-NCD is to be used to fabricate high quality devices, the problematic as-grown surface roughness must be corrected ,8. DespiAn alternative to the mechanical polishing of NCD films is chemical mechanical polishing (CMP), a technique commonly used in industrial integrated circuit fabrication processes. This technique replaces the cast iron scaife with a soft polyester-based pad and has recently been demonstrated to be effective in polishing both single-crystal diamond and NCD films ,23. WhilCD films  but the CD films . In this2. 2.1. in situ pyrometric interferometry to be 520 nm. This thickness was obtained after a deposition time of 150 min, after which the film was cooled in a hydrogen plasma and subsequently removed from the reactor chamber.The B-NCD film used for this study was grown using microwave plasma-assisted CVD . The sububstrate . The sub2.2. The resistive superconducting transition of the B-NCD film was measured before and after CMP processing. Whole wafers are required for the polishing sample holder, therefore the complete 22.3. in situ conditioning was used throughout to maintain the optimal surface texture of the polishing pad. The film was polished intermittently for 14 h in total, with The B-NCD film was polished by CMP using a Logitech Tribo polishing system equipped with a polyurethane impregnated polyester felt ) polishing pad with Logitech SF-1 Polishing Fluid – an alkaline colloidal silica polishing slurry. The polishing technique and proposed mechanisms are well described elsewhere ,23,27. P3. 3.1. Figure l growth . After pl growth ,27, therl growth .Corresponding AFM micrographs are shown in Figure 3.2. To assess the extent to which CMP affects the superconducting transition temperature, the variation of the normalised resistance with temperature is shown in Figure u et al.  which suFollowing the first six hours of polishing,  and ΔTC . Since C and ΔTC  that sug4. To conclude, it has been shown that a B-NCD film of as-grown surface roughness 44.0 nm RMS retains superconductivity following more than 10 h of chemical mechanical polishing, until a surface roughness of 1.5 nm RMS is reached. The observed


Four studies showed improvements in fasting blood glucose in low-GI diets compared to higher-GI diets or control: low-GI diet = 150.8 ± 8.7 vs. higher-GI diet = 157.8 ± 10.4 mg/dL, mean ± SD p = 0.43; low-GI diet = 127.7 vs. high-cereal fibre diet = 136.8 mg/dL, p = 0.02; low-GI diet = 6.5 (5.6–8.4) vs. standard diabetic diet = 6.7 (6.1–7.5) mmol/L, median and interquartile range p > 0.05; and low-GI diet = 7.3 ± 0.3 vs. conventional carbohydrate exchange diet = 7.7 ± 0.4 mmol/L, mean ± SEM (Standard Error of Mean) p < 0.05. The results of the meta-analysis and sensitivity tests demonstrated significant differences  between the low-GI diet and the higher-GI diet or control diet in relation to glycated haemoglobin. Differences between the low-GI diet and higher-GI diet or control were significant (p < 0.05) with respect to the fasting blood glucose following meta-analysis. Conclusion: The low-GI diet is more effective in controlling glycated haemoglobin and fasting blood glucose compared with a higher-GI diet or control in patients with type 2 diabetes.Background: The increasing prevalence of diabetes in the United Kingdom and worldwide calls for new approaches to its management, and diets with low glycaemic index have been proposed as a useful means for managing glucose response. However, there are conflicting reports and differences in the results of studies in terms of their effectiveness. Furthermore, the impact of low-glycaemic index diets and their long-term use in patients with type 2 diabetes remains unclear. Objectives: The objective of this study was to conduct a systematic review and meta-analysis of the effect of low-glycaemic index diets in patients with type 2 diabetes. Methods: Search methods: Randomised controlled studies were selected from a number of databases  based on the Population, Intervention, Comparator, Outcomes and Study designs (PICOS) framework. The search terms included synonyms and Medical Subject Headings (MeSH) and involved the use of Boolean operators (AND/OR) which allowed the combination of words and search terms. Selection criteria: As per the selection criteria, the following types of articles were selected: studies on randomised controlled trials, with year of publication between 2008 and 2018, including patients with type 2 diabetes. Thus, studies involving patients with gestational and type 1 diabetes were excluded, as were observational studies. Nine articles which met the inclusion criteria were selected for the systematic review, whereas only six articles which met the criteria were included in the meta-analysis. Data collection and analysis: Studies were evaluated for quality and risk of bias. In addition, heterogeneity, meta-analysis, and sensitivity tests of the extracted data were carried out using Review Manager 5.3 . Results: The findings of the systematic review showed that the low-glycaemic index (low-GI) diet resulted in a significant improvement (<0.05) in glycated haemoglobin (HbA1c) in two studies: low-GI diet Δ = −0.5%  vs. high-cereal fibre diet Δ = −0.18% ; and low-GI legume diet Δ = −0.5%  vs. high-wheat fibre diet Δ = −0.3% . There was a slight improvement in one study (low glycaemic response = 6.5% (6.3–7.1) vs. control = 6.6% (6.3–7.0) and no significant difference ( The increasing prevalence of diabetes and its impact on morbidity and mortality have become global problems ,2. AboutFoods that are composed of carbohydrates which break down quickly during the process of digestion (such as white bread) and that are rapidly absorbed into the blood stream are often termed as foods with high glycaemic index (GI) ,8,9. FooThe GI is a measure of the percentage of the area under curve (AUC) with respect to 2-h blood glucose following the ingestion of a test diet compared with a standard diet  . It can The GI value of food is not based on the characteristics of the individual that consumed it, instead, it depends on the food consumed ,14,15. TStrategies for managing diabetes often rely on lifestyle modifications, including dietary interventions and pharmacological approaches. There is also evidence that the consumption of diets with high glycaemic index and glycaemic load over a long period of time may have implications for metabolism and health, including chronic hyperglycaemia and hyperinsulinaemia, which can lead to insulin resistance and diabetes . In addiHowever, the current systematic review is based only on randomised controlled trials and involves only adults with type 2 diabetes, and the outcomes of interest are HbA1c and fasting blood glucose. In addition, there is currently no globally agreed form of diet for managing patients with diabetes . TherefoThis is a systematic review and meta-analysis which evaluates the effect of the low-glycaemic index diet in patients with type 2 diabetesResearch question: Is a low-GI diet effective in improving glycaemia in patients with type 2 diabetes compared with a higher-GI diet?Only studies involving randomised controlled trials were selected for this review .The participants in the studies selected were adult patients with type 2 diabetes .The effect of low-glycaemic index diet was compared with higher-glycaemic index diet or control  in adult patients with type 2 diabetes. The higher-GI or control diets were classified as having a higher glycaemic index based on the lower GI values of the intervention diets (low-GI diet).The following were the outcome measures of interest:Blood glucose parameters: Glycated haemoglobin (%), fasting blood glucose (mg/dL).The Population, Intervention, Comparator, Outcomes and Study designs (PICOS) framework was used to identify articles in the various databases ,25. The A number of research databases were used to search for relevant articles for this review. These included EBSCoHost research databases with links to Health Research databases which incorporate Academic Search Premier, Medline, the Psychology and Behavioural Sciences Collection, PSYCInfo, and the Cumulative Index to Nursing and Allied Health Literature (CINAHL) Plus. In addition, Pubmed was searched for useful articles .The Web of Science database which encompasses the BIOSIS citation index was searched for conference papers, and the reference list of articles were also searched.Only primary research on randomised controlled studies carried out between 2008 and 2018 were included in this review . This peIn addition, only studies involving adults with type 2 diabetes and the use of the dietary glycaemic index were included. Studies written in English from across the world have been included as diabetes is a worldwide problem.Therefore, other studies involving patients with type 1 diabetes or gestational diabetes and animal studies were excluded from this review . SimilarThe quality of the peer-reviewed articles was evaluated using the checklists for quantitative studies  and the Data from the selected articles were extracted separately by all the authors based on an agreed framework and verified by all the authors following completion. p value of 0.1 was used to determine statistical significance [I2 value was ˂50 and this indicated low heterogeneity for the studies included in both the glycated haemoglobin and fasting blood glucose analyses. Therefore, the fixed effects model was used for both the meta-analysis and the sensitivity tests.The meta-analysis of data was carried out using Review Manager (RevMan) 5.3 software . Changesificance . The hetA fail-safe number was also calculated for both outcomes of interest. A sensitivity analysis involving a repeat of the meta-analysis of studies that were definitely known to be eligible was also conducted . This prThe assessment tool used for evaluating the risk of bias was a domain-based evaluation tool . The proWith respect to the systematic review, only nine articles ,34,35,36The length of study ranged from 2 weeks to 22 months. In terms of the interventions, these involved comparing the low-GI diet with the higher-GI diet or control  . p < 0.05) lower values than the higher-GI diet or the control. The study by Jenkins et al. [p > 0.05) in one study [In most of the studies ,34,35,36s et al.  also shone study , while dne study ,33 althop < 0.05) in HbA1c levels in two studies [In studies reporting on the effect of dietary GI on the HbA1c levels, diets with low GI were shown to result in a significant improvement  100% of the studies showed low risk of bias . With rep < 0.001) and in the sensitivity test (p < 0.001) (removing the study with the most weight)  . p = 0.33) in the studies used to evaluate the effect of low-GI diet on glycated haemoglobin. The I2 test showed I2 = 13% for meta-analysis and I2 = 33% for the sensitivity test, again, confirming low heterogeneity of the studies included.There was low heterogeneity (Nfs0.05 = (∑Z/1.64)2 − S  to calculate the fail-safe number (Nfs0.05)24, the Nfs0.05 was 16. That is, another 16 negative studies would be needed to reverse this result, thus indicating that the result is stable.The result of the sensitivity test  showed tp < 0.05), this was not so in the sensitivity test (p = 0.15) (removing the study with the most weight) (Nfs0.05) with respect to fasting blood glucose was 5. With respect to evaluating the effect of the low-GI diet on fasting blood glucose compared to the higher-GI diet or control diet, the meta-analysis favoured the low-GI diet. However, while the differences were significant in the meta-analysis ( weight) . These s weight)  indicatep value (p = 0.36) and the chi-squared statistic (2.04) relative to the degree of freedom (2) would suggest that there was low heterogeneity between the studies. This was confirmed by the results of the I2 test. The heterogeneity test for fasting blood glucose showed I2 = 2% for meta-analysis and I2 = 45% for the sensitivity test, indicating low heterogeneity of the studies included.Regarding the heterogeneity test, the high Based on the findings of the systematic review, the low-GI diet resulted in greater improvement in glycated haemoglobin ,30 and fThere was no statistically significant difference between the low-GI and higher-GI diets in relation to HbA1c and fasting blood glucose in the study by Visek et al. . Acute gThe Food and Agricultural Organisation  has recoThis review used a systematic approach and meta-analysis to provide contemporary evidence on the positive effects of low glycaemic index diet on fasting blood glucose and glycated haemoglobin. The limitation is in the number of studies included in the meta-analysis. In addition, most of the studies had relatively small sample sizes. The availability and inclusion of more studies would have increased its wider application.p < 0.001 and p < 0.001, respectively) between low-GI diets and higher-GI diets or control diets in relation to glycated haemoglobin. In addition, differences between the low-GI diet and the higher-GI diet or control were significant (p < 0.05) with respect to the fasting blood glucose following meta-analysis. The findings of this systematic review and meta-analysis have shown that low-GI diets are more effective in controlling HbA1c and fasting blood glucose compared with higher-GI diets or control diets in patients with type 2 diabetes. Although the outcomes of the individual studies were sometimes different with respect to the variables of interest, the results of the meta-analysis and sensitivity tests have demonstrated significant differences (It will useful to evaluate the long-term effectiveness of low-glycaemic index diet in patients with type 2 diabetes.


Recilia dorsalis. In this study, the transmission characteristics of RSMV by R. dorsalis were investigated. Under suitable growth conditions for R. dorsalis, the RSMV acquisition rate reached 71.9% in the second-generation population raised on RSMV-infected rice plants. The minimum acquisition and inoculation access periods of R. dorsalis were 3 and 30 min, respectively. The minimum and maximum latent transmission periods of RSMV in R. dorsalis were 6 and 18 d, respectively, and some R. dorsalis intermittently transmitted RSMV at 2–6 d intervals. Our findings revealed that the virus can replicate in the leafhopper body, but is likely not transovarially transmitted to offspring. These transmission characteristics will help guide the formulation of RSMV prevention and control strategies.Rice stripe mosaic virus (RSMV) is a newly discovered species of cytorhabdovirus infecting rice plants that is transmitted by the leafhopper  Oryza sativa) accounts for the majority of the world's cereal food production. Viral diseases are serious threats to rice production  and Rice tungro spherical virus  in the Philippines and Indonesian  and Rice black streaked dwarf virus  in China and Japan  in China and Southeast Asia  as well as a new species Southern rice black-streaked dwarf virus  in China and Vietnam , which occurs in southern China  used in this study were grown as previously described . From this population, one pair  were mated and reared on one healthy 3-leaf stage rice seedling that was replaced daily. Then, their offspring and the rice seedlings used to rear the insects were again tested by RT-PCR to confirm they were RSMV negative. The insects were propagated for over three generations before being used. The virus was transmitted by the leafhopper R. dorsalis, and maintained on rice plants growing in insect-proof greenhouses. The leafhoppers were maintained in insect-proof cages at 25°C and 80% relative humidity under a 16-h light/8-h dark photoperiod.The rice plants . RT-PCR amplification of the viral RNA was carried out using a One-Step RNA PCR kit . The virus-specific detection primers and PCR conditions were similar to those previously reported  and maintained for 15 d for virus infection and propagation. The insects that were still alive after 15 d were collected and tested for the presence of the virus using RT-PCR. The viral acquisition access period of R. dorsalis was calculated from the RT-PCR results.The RSMV-negative R. dorsalis 3rd–4th instar nymphs or adults propagated on RSMV-infected rice plants were collected, starved for 4 h, and then transferred to the healthy 3-leaf stage rice seedlings. The insects were allowed to feed on the seedlings for 3, 5, 10, 30, or 60 min, and were then individually tested using RT-PCR to determine whether they were RSMV positive. The seedlings inoculated with confirmed virus-positive leafhoppers were grown for 15 d. The inoculation access period of the leafhopper was determined from the results of the RT-PCR detection of the tested plants at 15 d after inoculation.The second-generation R. dorsalis, the method reported by Pu et al. . The tested plants were replaced by healthy ones every 2 d until the insects died. Then, the tested plants were grown in an insect-proof greenhouse, and RT-PCR was conducted after 10 d. The latent period of RSMV in leafhopper was calculated from the RT-PCR results.To determine the latent period of RSMV in u et al.  was follR. dorsalis adults propagated on RSMV-infected rice plants were collected, and 70 pairs  were transferred to healthy 3-leaf stage rice seedlings in glass culture tubes (one pair per tube). They were kept for 12 d and the seedlings were replaced daily. After the eggs were laid, the adults were individually tested by RT-PCR to determine whether they were RSMV positive. The eggs produced by females that were RSMV positive were transferred to healthy rice seedlings until they hatched, then the 3rd–4th instar nymphs were tested for RSMV.To determine whether RSMV can be transovarially transmitted, the method reported by Chen et al.  was follR. dorsalis adults reared on RSMV-infected rice plants for more than 10 d were collected and their midgut tissues obtained. The tissues were excised on an ultra-microtome and fixed in 4% glutaraldehyde in cacodylate buffer (pH 7.4) at 4°C for 24 h, and then in 1% osmium tetroxide overnight. The fixed tissues were dehydrated through an alcohol series and embedded in Spurr's resin stained with 1% uranyl acetate and lead citrate  from the insect was reverse-transcribed using the AMV RNA PCR Kit (Takara) in a 10-μL reaction mixture volume. The cDNA was diluted 10 times according to the manufacturer's protocol , and each qPCR reaction consisted of 2 μL cDNA, 0.5 μL of each primer (10 μM), 12.5 μL of SYBR®Premix Ex Taq™ II in a total volume of 20 μL. The PCR parameters consisted of one cycle at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. RT-qPCR reactions were carried out in a CFX96 Touch™ real-time PCR detection system . The RSMV-specific primers N-F (5′-TAGAAGGGCGGCTACCTCA-3′) and N-R (5′-GACAGCCACATAGCGGAGAA-3′) were used for the viral content's relative quantification, and the R. dorsalis' Actin gene, with the primers Actin-F (5′-AGAAGTCCTACGAGTTGCCTGATG-3′) and Actin-R (5′-TTCATGATGGAGTTGTAGACGGTC-3′), was used as a reference , 80.0% (24/30), and 69.0% (20/29) of R. dorsalis adults were RSMV positive. The values were not significantly different. This indicated that R. dorsalis has a high frequency of virus acquisition from RSMV-infected rice plants after long-term feeding, and its viral mean acquisition rate can reach 71.9% (64/89).In the three experiments in which RSMV was detected in R. dorsalis nymphs and adults could become RSMV positive after feeding on the virus-infected rice plants for 3 min, with acquisition rates of 24.4 and 19.2%, respectively  and the maximum period was 18 d (in insect no. 21). The latent period averaged 13 d (ranging from 6 to 18 d) under our experimental conditions. Some of the tested individuals  could continuously transmit RSMV throughout their lives, but most of them intermittently transmitted the virus at intervals of 2–6 d. Three tested individuals (nos. 1–3) could not transmit the virus at any time during their lifespan.Using RT-PCR, 21 of the 32 tested leafhoppers were confirmed RSMV positive. The latent period of the virus in these 21 individuals was examined using viral transmission assays , that were absent in the negative leafhoppers  is mostly transmitted, with a high efficiency, by the small brown planthopper (Laodelphax striatellus), while another planthopper, Javesella pellucida, has a much lower transmission efficiency  is transmitted by the leafhopper Endria inimical, while Elymana virescens, which is a close relative, only acts as a secondary transmission vector , but the former is the primary transmission vector . Whether N. cincticeps is an additional vector of RSMV needs further investigation.Plant rhabdoviruses have a high degree of vector specificity and most are transmitted naturally by one or only a few closely related insect species , can be transovarially transmitted to offspring of the common vector R. dorsalis , L. striatellus-transmitted BYSMV and R. dorsalis-transmitted RGDV  in N. cincticeps and SRBSDV in Sogatella furcifera (Chiu et al., The latent period is required for the persistent-propagative transmission of viruses and is different lengths in different vector–virus combinations (Ammar et al.,  d Table , which iR. dorsalis to transmit RSMV to rice plants, and most needed 60 min of feeding (Table R. dorsalis transmitting RSMV was longer than that of E. inimical when transmitting WASMV, which takes only 15 min (Slykhuis, G. sonora transmitting SSMV, which takes 1 h (Creamer et al., R. dorsalis transmitting RSMV was longer than that of R. dorsalis when transmitting RDV, which takes only 10 min (Shinkai, S. furcifera transmitting SRBSDV, which takes 30 min (Pu et al., At least 30 min of feeding was needed for ng Table . CompareShinkai, , but wasSimilar to most persistent-propagative transmission viruses (Creamer et al., The vector-mediated transmission of plant viruses is fascinating biologically and evolutionarily. However, there is little information available on genes or other factors involved in RSMV transmission. The rhabdoviruses encode glycoprotein, which is exposed to the surfaces of the viral particles and is essential for virus attachment to cell receptors (Jackson et al., GZ conceived and designed the experiments. XY performed the experiments and wrote the draft. TZ analyzed the data and revised the manuscript. BC prepared the materials. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P < 0.001), with an AUC of 0.721 (95% CI: 0.628–0.817). In patients with tumors larger than 2 cm (n = 40) and those with clear-cell histology (n = 44), the AUCs for ESM-1 were 0.771 and 0.721, respectively. In control subjects, serum ESM-1 levels were higher in older (>50 years) individuals (P < 0.001). Among the study cohort, the AUCs for ESM-1 were 0.813 in individuals 50 years of age or younger (n = 55) and 0.637 in individuals older than 50 years (n = 57). In RCC patients, serum ESM-1 levels were reduced 1 month (P = 0.047) and 3 months (P = 0.009) after surgery. These results suggest serum ESM-1 can serve as a serologic biomarker for diagnosing and monitoring RCC, particularly in patients younger than 50 years.To determine the suitability of serum endocan (ESM-1) levels for diagnosing and monitoring renal cell carcinoma (RCC), we measure serum ESM-1 levels in 56 RCC patients who had undergone radical or partial nephrectomies and 56 age- and sex-matched healthy kidney donors. Measurements were made before and 1 month and 3 months after surgery. The areas under the curve (AUCs) were determined from receiver operating characteristic (ROC) analyses. RCC patients had higher mean serum ESM-1 levels than control subjects (0.59 ± 0.07 vs. 0.52 ± 0.08 ng/mL,  Renal cell carcinoma (RCC) is the most common malignant tumor in the kidney . Althouget al. [Endocan, or endothelial cell-specific molecule-1 (ESM-1), is a soluble 50-kDa dermatan sulfate proteoglycan expressed by the vascular endothelium . ESM-1 iet al.  suggesteP = 0.027). In the control group, the mean serum ESM-1 level did not vary according to sex  but was higher in older (>50 years) participants than in participants who were younger (≤50 years) . In the RCC group, the mean serum ESM-1 levels were not different between patients with clear-cell RCC and non–clear-cell RCC. In patients with clear-cell RCC, the serum ESM-1 levels were significantly higher in patients with large tumors and higher stages. However, no statistically significant differences were seen among patients in the entire RCC group (Table Patient characteristics are summarized in Table P < 0.001)  and with clear-cell histology (n = 44), AUCs for ESM-1 levels were 0.771 and 0.721, respectively. Because of the older age of patients in the RCC group and the higher levels of serum ESM-1 in older participants, we performed a multivariate linear regression analysis. After controlling for effects of age and gender, we found that RCC significantly affects the serum level of ESM-1 (P < 0.001) .Patients with RCC had higher serum ESM-1 levels than control group participants  Figure . The are) Figure . With a 1) Table .When we In this study, we found that serum ESM-1 levels were higher in patients with RCC than in healthy individuals. Although serum ESM-1 levels were influenced by age, they were also promoted by the presence of RCC. Thus, serum ESM-1 can potentially be used as a diagnostic marker for RCC.et al. [Increased levels of ESM-1 have been found in the sera of patients with colorectal cancer, hepatocellular carcinoma, and acute leukemia –15. Moreet al.  demonstret al. . Howeveret al. . MoreoveIn the results of the subgroup analysis, we found that the diagnostic performance of serum ESM-1 for RCC improved in patients with tumor size larger than 2 cm. It is attributable to the reflection of the tumor burden by serum ESM-1, which is in line with the previous study . With tht-tests. The decreased levels were maintained for up to 3 months postoperatively. We believe these findings suggests that serum ESM-1 can be a surveillance marker for RCC.The measurement of serum ESM-1 levels after the surgical removal of RCC was not performed in previous studies. We found that the levels decreased postoperatively, although they remained higher than in the control group. We could not exclude an effect from the surgery itself, because postoperative serum ESM-1 levels were not obtained from the healthy control subjects. Nevertheless, the differences were statistically significant in paired Although elevated serum levels of ESM-1 in RCC have been reported, this study included the largest number of patients with RCC and healthy control subjects. Our results indicated that ESM-1 can be used as both a diagnostic and a surveillance biomarker. In addition, we found that ESM-1 showed improved diagnostic performance in a young cohort. These novel findings represent strengths of our study.Some limitations of this study must be considered: First, this study did not include patients with benign renal masses. Currently, we only have a small number of patients with benign renal masses in our database, and a larger cohort is needed to investigate the efficacy of serum ESM-1 levels in differentiating RCC from benign renal masses. Second, serum ESM-1 levels are also elevated in other types of cancers and thus are not specific for RCC. However, unlike hepatocellular carcinoma and colorectal cancer, RCC has no serologic biomarker for diagnosis. Third, the prognostic ability of ESM-1 in RCC was not assessed in this study, because only two cases of recurrence were seen during the median 27 months of follow-up. Therefore, a longer follow-up involving patients with more advanced stages of RCC is required to evaluate the use of serum ESM-1 levels for prognosis.The results of our study show that serum ESM-1 levels can serve as a diagnostic biomarker for RCC, particularly in patients younger than 50 years. Moreover, the assessment of serum ESM-1 levels can be utilized for monitoring patients with RCC after surgery. Although a validation in a larger cohort is necessary, we conclude that serum ESM-1 can potentially be clinically applied for diagnosing and monitoring RCC.From October 2013 to March 2015, we prospectively enrolled patients undergoing nephrectomies in the urology department at Severance Hospital. In total, 56 patients with pathologically confirmed RCC underwent partial or radical nephrectomies. For the control group, we included 56 age-matched and sex-matched individuals from a cohort of healthy kidney donors who had undergone donor nephrectomies during the same period. This study was approved by the Institutional Review Board of Severance Hospital (IRB no. 4-2013-0166), and informed consent was obtained from all participants before study enrollment.g within 30 minutes of collection, and the sera were divided into aliquots and stored at −80°C for subsequent measurements of ESM-1 levels.Blood sampling was performed the day before surgery for all participants and at 1 month and 3 months after surgery for patients in the RCC group. Blood samples were centrifuged for 15 minutes at 1,000 × 2SO4. The absorbance was determined at 450 nm on a spectrophotometer. All assays were performed in duplicate.In all serum samples, the level of ESM-1 was quantified by use of a commercially available ELISA kit . Serum was diluted twofold before analysis. Experiments were performed according to manufacturer’s instructions. Microwell plates were coated with 100 μL of capture antibody (2 μg/mL) and incubated overnight at 4°C. The coated plates were blocked with 5% skim milk in tris buffered saline (TBS) for 1 hour at 37°C. After a washing step with phosphate buffered saline (PBS), the plates were incubated with 100 μL of serum specimen for 1 hour, and then washed and incubated with 100 μL of secondary antibody  for 1 hour at room temperature. After the washing, 100 μL of streptavidin-horseradish peroxidase  was added and incubated for 30 minutes. After a final washing, 50 μL of tetramethylbenzidine substrate was added and the reaction was stopped with 1 N HESM-1 levels in the control group were compared according to the sex and age (≤50 years vs. >50 years) of the participants. Serum ESM-1 levels were then compared between control subjects and RCC patients, as well as according to tumor size, pathologic stage, Fuhrman grade, and histology in RCC patients. ROC curves were generated and the AUCs were calculated. The optimal cutoff value, which maximizes sensitivity and specificity, was obtained by use of the Youden index. In addition, we performed subgroup analyses of RCC patients with tumor sizes larger than 2 cm or with clear-cell histology.Although RCC and control groups were matched by age and sex, patients in the RCC group were older than those in the control group. Because of the higher levels of serum ESM-1 in older participants than in their counterparts in the control group, we performed multivariate linear regression analyses to evaluate whether the presence of RCC significantly increases the level of serum ESM-1.t-tests or one-way analyses of variance, whereas qualitative variables were compared by use of chi-square or Fisher’s exact tests. Statistical analyses were performed by the Statistical Package for Social Science for Windows, Version 18.0 . A P-value < 0.05 was considered significant, and all P-values were two-sided. The ROC analyses and plotting graphics were performed by R Version 3.2.5 software (http://www.r-project.org).Preoperative and postoperative (at 1 month and 3 months) ESM-1 levels were compared. The quantitative values were compared by use of Student’s


Carbohydrates represent an important fraction of labile and semi-labile marine organic matter that is mainly comprised of exopolymeric substances derived from phytoplankton exudation and decay. This study investigates the composition of total combined carbohydrates  and the community development of free-living (0.2–3 μm) and particle-associated (PA) (3–10 μm) bacterioplankton during a spring phytoplankton bloom in the southern North Sea. Furthermore, rates were determined for the extracellular enzymatic hydrolysis that catalyzes the initial step in bacterial organic matter remineralization. Concentrations of tCCHO greatly increased during bloom development, while the composition showed only minor changes over time. The combined concentration of glucose, galactose, fucose, rhamnose, galactosamine, glucosamine, and glucuronic acid in tCCHO was a significant factor shaping the community composition of the PA bacteria. The richness of PA bacteria greatly increased in the post-bloom phase. At the same time, the increase in extracellular β-glucosidase activity was sufficient to explain the observed decrease in tCCHO, indicating the efficient utilization of carbohydrates by the bacterioplankton community during the post-bloom phase. Our results suggest that carbohydrate concentration and composition are important factors in the multifactorial environmental control of bacterioplankton succession and the enzymatic hydrolysis of organic matter during phytoplankton blooms. In seasonal seas, phytoplankton development and temperature have repeatedly been reported to structure bacterioplankton communities  composition is only poorly resolved. Numerous compounds in the vast pool of marine OM may support species-rich bacterioplankton communities, which are, in turn, able to efficiently remineralize large portions of the OM. In laboratory experiments, bacterioplankton exhibit specific OM uptake at a class and clade level . Single bacteria .While large portions of marine OM are still chemically uncharacterized, carbohydrates have been identified as the largest fraction of characterized marine OM . Recent Bacteria can take up LMW compounds as large as 0.6–0.8 kDa through porins and slightly larger compounds via TonB-dependent transporters  to meet Understanding the interaction between bacterioplankton and diverse OM is important for our understanding of biogeochemistry . It is ain situ meta-transcriptomics. It investigates the influence of carbohydrate composition and concentration in seawater on the community structure of free-living (FL) and particle-associated (PA) bacteria by combining, for the first time, a detailed analysis of the composition of total combined carbohydrates  using high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) and bacterial community analysis using Automated Ribosomal Intergenic Spacer Analysis (ARISA) and Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization (CARD-FISH). Furthermore, it investigates the extracellular enzymatic activity of the bacterial community in relation to carbohydrates and the wide range of physicochemical and biological factors at the transition from winter to spring in the temperate North Sea.This field study aims to fill the gap between laboratory studies of single bacterial groups or carbohydrates and results inferred from Surface water (∼1 m depth) was collected in a 10-L plastic carboy in the morning twice a week from February 2nd to May 18th 2010 from a research vessel at the long-term North Sea monitoring station “Helgoland Roads” between the islands of Helgoland  in the German Bight, North Sea see .After pre-filtration through 10 μm-pore size filters, PA bacteria were collected by filtering 2 L of seawater through 3-μm filters . To collect FL bacteria, 500 mL of the resulting filtrate was filtered through a 0.2-μm filter . All filters were cut into three equal pieces using sterilized pincers and scalpel and stored in a 2 mL-reaction tube at -20°C until processing.The DNA from all three filter pieces was extracted simultaneously using lysozyme followed by phenol-chloroform-isoamylalcohol purification , was applied. The wells were loaded with 0.25 or 0.5 μL of samples, depending on the amount needed to visualize bands clearly. Values obtained from the application of a 0.5 μL sample were divided by two prior to data analysis. The volume of applied sample was confirmed to have no significant effect on Sørensen-similarities in the PA-  and FL-  fractions. The software package BioNumerics  was used to analyze the gel images. The bands with a length between 300 and 1500 bp were binned into size classes (termed “ARISA-band-classes”) to correct for minor length variations of the IGS regions between lineages with almost identical 16S rDNA-sequences. Bins of 3 bp were used for fragments up to 700 bp in length, bins of 5 bp were used for fragments between 700 and 1,000 bp, and bins of 10 bp were used for fragments larger than 1,000 bp , as described in 1,000 bp . In thisIn Situ Hybridization (CARD-FISH) of non-prefiltered samples on 0.2-μm filters were taken from Values for Catalyzed Reporter Deposition Fluorescence 1  as part of the Helgoland Roads LTER time series, which is also accessible via the open database PANGEA1 . Surface1 . Salinit1 . A fluortrations . Inorgantmeasured is the temperature in °C, measured simultaneously with pH using a combined electrode  calibrated with standard buffer solutions .where Particulate carbon was collected on pre-combusted GF/F filters , which were stored at -20°C until analysis. The filtration volume (80–380 mL) was adjusted to the particle loading of the seawater. Filters were treated with 200 μL of carbon-free HCl (0.2 N) to remove inorganic carbon and dried at 60°C for approximately 48 h. After drying, filters were packed into small Zn-cartridges and analyzed using an elemental analyzer .For analysis of DOC concentration, seawater was filtered through combusted  GF/F filters , and 15 mL was sealed in combusted glass ampoules. Samples were stored at -80°C in the dark. Some of the ampoules were damaged during transport to the mainland, resulting in reduced temporal resolution. Acidified samples  were analyzed using high-temperature catalytic oxidation  on a ShiTotal organic carbon (TOC) was calculated as the sum of particulate organic carbon (POC) and DOC.2), were analyzed on a Dionex ICS3000 system combining high performance anion exchange chromatography (HPAEC) and pulsed amperometric detection (PAD) using a Dionex CarboPac PA10 analytical column (2 × 250 mm) coupled to a Dionex CarboPac PA10 guard column (2 × 50 mm). The autosampler (Dionex AS50) was maintained at 8°C, and 17.5μL of sample was injected for each analysis. After every second sample, 17.5 μL of a mixed sugar solution was injected for standardization. Blanks  were analyzed using the same procedure used for the samples and subtracted from sample concentration. We identified 12 carbohydrate monomers (Supplementary Table S1). The neutral sugars arabinose (Ara), fucose (Fuc), galactose , glucose (Glc), and rhamnose (Rha) were investigated, as well as the co-eluting sugars mannose and xylose (Man/Xyl). The acidic sugars galacturonic acid , gluconic acid (GlcA), glucuronic acid (GlcUA) and muramic acid (Mur) were measured. In addition, the concentrations of the two amino sugars galactosamine  and glucosamine (GlcN) were determined. The detection limit for the method was 1 nmol L-1. Values for all sugar components and standard deviations can be found in Supplementary Table S2.Analysis of total combined carbohydrates , which are a portion of high molecular weight organic matter (HMW-OM), was performed according to L-leucyl-4-methylcoum-arinylamid-hydrochlorid and 4-methylumbelliferyl-phosphate, respectively. The substrate analogs were added to seawater samples at final concentrations of 1, 5, 10, 20, 50, 80, 100, and 200 μmol L-1 to determine enzyme kinetics. To measure fluorescence, the infinite M200 plate reader  was used. The fluorescence emitted by 4-methylumbelliferone (MUF) was detected at 365 nm excitation and 440 nm emission wavelengths, and that of 7-amino-4-methyl-coumarine (AMC) was detected at 380 nm excitation and 440 nm emission wavelengths. Fluorescence units were converted into concentrations of MUF or AMC after calibration with standard solutions. Enzymatic rates were calculated from the increase in MUF or AMC concentration, over time. An initial fluorescence measurement was conducted immediately after the addition of the substrate analog, followed by two measurements within 4 h of incubation in the dark at the approximate in situ temperature for the sampling day (1–8°C). The initial fluorescence was subtracted as background fluorescence from the fluorescence measured at each time point. The slope of the linear regression between incubation time and the concentration of the fluorescent marker was applied for rate calculations. Experimental data were fitted using the Michaelis–Menten equation to determine the maximum velocity (Vmax) of the enzymatic reactions. To estimate the amount of substrate hydrolyzed by β-glucosidase (β-Glcase) during the post-bloom phase, we calculated the enzymatic rate for each day using a mean post-bloom concentration of 2.13 μmol L-1 Glc. The resulting mean rate (83.05 nmol L-1 d-1) was multiplied by the number of days (26) in the post-bloom phase.The activity of extracellular enzymes was determined by the use of fluorogenic substrate analogs . The ratr > 0.8) Fuc, Rha, Gal, Glc, GalN, GlcN, and GlcUA were pooled.For multivariate statistical analyses, the software package PRIMER v.6 and the add-on PERMANOVA+  were used. The analyses were performed using Sørensen-similarity-matrices generated from square-root transformed ARISA-band-class data. To test for community assemblage differences between phytoplankton bloom phases, permutational multivariate analysis of variance (PERMANOVA) was applied. Distance-based multivariate multiple regression (DistLM) was used to calculate correlations of community composition to environmental factors, and distance-based redundancy analysis (dbRDA) was used to visualize these correlations. For this analysis, the highly collinear combined carbohydrates  was used to determine the state of the phytoplankton bloom. After low Chl a concentrations (<1 μg L-1) in February, a diatom-dominated spring phytoplankton bloom developed at the long-term monitoring station “Helgoland Roads” . Bloom development started in mid-March and reached Chl a values of up to 12 μg L-1 in April . The onset of the bloom coincided with a strong peak in dissolved inorganic phosphate (DIP) concentration and a slight increase in sea surface temperature . The bloom development was accompanied by rising seawater pH. Salinity decreased from approximately 34 in February to approximately 31 at the end of April . The phytoplankton bloom terminated in late April, likely due to the depletion of DIP and silicate (SiO2) .Chlorophyll -1 , while DOC showed a maximum value of 151 μmol L-1 at the peak of the bloom. TOC values were calculated as the sum of POC and DOC and reached maximum values of 135 and 187 μmol L-1 before and after the phytoplankton bloom, respectively. Bloom development resulted in the accumulation of tCCHO, which contributed up to 14% of TOC in the post-bloom phase . The subsequent decline in tCCHO and POC lagged behind the decrease in diatom cell numbers and Chl a . The carbon contribution of tCCHO to TOC strongly increased during the bloom . Several carbohydrates in HMW-OM showed very similar temporal dynamics (Pearson r > 0.8) during spring 2010. Therefore, for the statistical analysis, glucose, galactose, fucose, rhamnose, galactosamine, glucosamine, and glucuronic acid were pooled into a single factor and designated as collinear CHO (colCHO). Its concentration strongly increased toward the end of the phytoplankton boom and maintained high values during the early post-bloom phase . In contrast, Ara showed concentrations near the detection limit during the entire study, while GalUA revealed strong dynamics unrelated to the phytoplankton bloom. Throughout the bloom, the composition of tCCHO was dominated by neutral sugars (60–90 mol%). Glc alone contributed an average 53 mol%, with maximum concentrations of 3 μmol monomer equivalent L-1 in the late bloom and post-bloom phase . Concentrations of Man/Xyl, Fuc and Gal increased with the onset of the phytoplankton bloom and maintained high values during the post-bloom phase, though maximum concentrations of 0.2–0.4 μmol L-1 were approximately an order of magnitude lower than that of Glc . Man/Xyl and Fuc showed similar mean concentrations of 0.13 and 0.16 μmol monomer equivalent L-1, respectively. In the late post-bloom period, Man/Xyl strongly increased, while Fuc decreased rapidly. Rha and Ara showed comparatively low concentrations ≤ 0.1 μmol L-1 throughout the investigation period.During the bloom, POC increased with diatom abundance, reaching a maximum of 54 μmol LFigure 2C). Concentrations increased again until the end of the phytoplankton bloom, reaching values of 0.2–0.3 μmol L-1, comparable to the concentrations of most neutral sugars, in April . The other acidic sugars GlcA, Mur, and GlcUA were only present in very low concentrations (below 0.02 μmol L-1). Of those, only GlcUA increased slightly in concentration toward the end of the bloom and maintained an elevated level during the post-bloom. Amino sugars were only present at low concentrations (below 0.1 μmol L-1) . However, both, GalN and GlcN concentrations increased toward the end of the bloom, with GlcN reaching values of up to 0.07–0.08 μmol L-1, higher than the concentrations of most acidic sugars during the late bloom and post-bloom phases.The acidic sugar GalUA was a major component of tCCHO before the onset of the bloom in February, followed by strongly declining concentrations in March  but were similar during the pre-bloom  and bloom phases . Both, PA and FL bacterial community significantly changed between phytoplankton bloom phases , revealing distinct temporal successions. DistLM shows that the development of PA communities is significantly related to three environmental variables. Apart from an abrupt change along a POC gradient  at the onset of the phytoplankton bloom, PA bacteria showed the strongest community development during the post-bloom phase, following a gradient of temperature (explaining ∼20%) and carbohydrates  . The FL bacterial community composition strongly changed during the ongoing phytoplankton bloom and during the post-bloom phase, mainly tracking with diatom abundance (∼14%) and temperature (∼34%), respectively . In total, significant environmental factors explained ∼60 and ∼48% of the variability in PA and FL communities, respectively. The richness of PA bacteria increased with rising Chl a and tCCHO concentrations, revealing a linear correlation  between colCHO concentration and PA bacterial richness . The PA richness reached a maximum approximately 1.5 weeks after the breakdown of the bloom at persistently high concentrations of tCCHO and was followed by a steep decrease in both richness and tCCHO thereafter . In contrast, the richness of FL bacteria did not resemble the temporal development of Chl a or diatom cell numbers during the bloom .Particle-associated and FL bacterial communities were significantly different during the post-bloom phase  showed a direct positive correlation to colCHO and to GalUA (Supplementary Table S3). Notably, these also correlated to temperature and pH, as well as POC and Chl a. Furthermore, they were correlated positively to all investigated extracellular enzymes. Although in total, Roseobacter did not correlate to any investigated parameter, the SAR11 subgroup correlated to pH and silicate. Bacteroidetes correlated to Chl a.Of the investigated microbial clades (CARD-FISH), only GAM42 . With the onset of the phytoplankton bloom, leucine-aminopeptidase (LAPase) activity increased and maintained high levels during the bloom, reaching a maximum of 0.95 μmol L-1 h-1 in the post-bloom phase. The activity of β-glucosidase (β-Glcase) increased toward the end of the bloom, concurrent with a strong increase in tCCHO concentrations . A maximum rate of 0.08 μmol L-1 h-1 was determined on May 11th. The amount of substrate hydrolyzed by β-Glcase during the post-bloom phase was estimated as approximately 2.16 μmol L-1, equivalent to approximately 72% of the total combined glucose that accumulated during the bloom . The rates of extracellular phosphatase (phos), increased rapidly after phosphate depletion  at the end of the bloom, indicating the enhanced use of organic phosphorous by bacteria and phosphatase-releasing phytoplankton groups. The co-incidence of phosphate depletion with a pronounced increase in phosphatase activity correlates well with the increase in phosphate transporters, as reported in The activity of extracellular enzymes was near the detection limit from February to mid-March (Table 2). Phos was positively correlated to DOC. In general, all neutral tCCHO except Ara and Man/Xyl showed strong (rs > 0.7) to very strong (rs > 0.9) positive correlations with LAPase, Glcase, and phos. Furthermore, amino sugars correlated well with enzyme activities. β-Glcase and LAPase activity were very strongly (rs > 0.9) correlated with Fuc, while LAPase was correlated very strongly (rs > 0.9) with Gal. Man/Xyl correlated with only amino-peptidase and phos, but not β-Glcase. The amino-sugars correlated well with enzyme activities. Glc-URA was the only acidic sugar showing a significant correlation with enzyme activities.All enzyme activities were positively correlated with seawater pH, temperature and POC but were negatively correlated to nutrients  were positively correlated to colCHO and showed higher abundances toward the end of the bloom. Further correlations of Gammaproteobacteria with POC and Chl a underline the importance of fresh OM derived from phytoplankton production for the growth of this group, which is a prominent member of bacterial communities associated with marine particles . In addition, approximately 75% of PA ARISA band classes did not correlate with the development of carbohydrate concentrations in any bloom phase. This might be explained by the fact that carbohydrates analyzed in this study are >1 kDa and thus part of the HMW-OM. The most labile fraction of freshly produced OM includes LMW compounds, which were shown to strongly affect bacterial community composition in in vitro experiments . In addition, the high activity of extracellular β-Glcase was observed well after the Chl a and tCCHO peaks, when PA bacterial richness was high. This indicates that for β-Glcase, high PA bacterial richness facilitates the enzymatic turnover of tCCHO. It is likely that the richer PA bacterial community produces not only high amounts of β-Glcase but also a high diversity of β-Glcase iso-enzymes  for laminarin and alginate that comprised genes of surface-exposed proteins such as oligomer transporters, substrate-binding proteins, and carbohydrate-active enzymes .The present study shows a correlation between the concentration of combined carbohydrates in HMW-OM and the diversity of PA bacteria during the development of a spring bloom, suggesting that the availability of carbohydrates contributes to the multifactorial control of marine bacterioplankton communities. Our study is in line with earlier findings demonstrating the importance of PA bacteria for total community activity . This himunities . In the es (pCO2 . Increaslability . CombiniMS developed the study concept and conducted the sampling as well as most of the sample- and data analysis. He also wrote the manuscript. JP helped develop the study concept, provided methodological support, especially with acquisition and analysis of extracellular enzyme data, and contributed to the manuscript. AE and AW provided lab space and financial and methodological support, helped develop the study concept and contributed to the manuscript. KW provided phytoplankton- and physicochemical data and contributed to the manuscript. JN provided data on dissolved organic carbon and contributed to the manuscript. GG provided lab space and financial and methodological support, helped develop the study concept, supported the statistical analysis and contributed to the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Treponema pallidum (TP) infections are considered classic transfusion-transmissible infections (TTIs). Few data are available about the prevalence of TTIs in patients before blood transfusion in China. This study aimed to investigate the seroprevalence of four TTIs among patients before blood transfusion in Xiangya Hospital Central South University, China.Human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and From 2011 to 2016, 442,121 hospitalized patients before possible blood transfusion were tested for hepatitis B surface antigen (HBsAg), anti-HCV, syphilis antibody (anti-TP) and anti-HIV.Of the 442,121 patients, the overall positivity of the four TTI serum markers was 15.35%. The positive rates of HBsAg, anti-HCV, anti-HIV and anti-TP were 10.98, 1.43, 0.16 and 2.78%, respectively. TTI serum markers showed a significant difference by gender, with positive rates of 17.98% for males and 12.79% for females. The prevalence of TTI serum markers varied significantly by age. The overall co-infection rate was 0.63%, and the top three multiple infections were HBV-TP, HBV-HCV, and HCV-TP. The co-infection rates of HBV-TP and HBV-HCV showed a significant decrease from 2011 to 2016, while the rates of other co-infections remained stable.The prevalence of TTIs in patients before blood transfusion is much higher compared to that in blood donors in the region. The infection rates of HIV and TP increased, and the infection rate of HBsAg decreased in recent years.The online version of this article (10.1186/s12879-018-3051-7) contains supplementary material, which is available to authorized users. Treponema pallidum (TP) are especially concerning because of their prolonged presence in the blood and body fluids of carriers [Blood transfusion, especially component transfusion such as packed red blood cells, platelet concentrates, fresh frozen plasma, individual factor concentrates, and cryoprecipitate, has become an indispensable medical treatment for the rescue of patients. However, blood transfusion still has some limitations, and the most significant one is the potential risk of transfusion-transmitted infections (TTIs) , 2. The carriers –4.With a general population infection rate of nearly 10.00% , the preThe prevalence of TTIs varies greatly in different areas and populations. For instance, in western China, the prevalence rate of HIV infection in donors was 0.31% , 13. WhiIn the present study, we analyzed the results of tests on patients before possible blood transfusion at a comprehensive teaching hospital in the central and southern region of China and evaluated the positivity of HBsAg, anti-HCV, anti-HIV and syphilis antibodies over a 6-year period. This study provides data about the prevalence and trends of these four TTIs in a large patient population.The cross-sectional study was approved by “Medical Ethics Committee of the Xiangya Hospital of Centre South University” in the year 2010. Written informed consent was obtained from all the patients to allow the use of anonymized test results of their blood samples. Consent was obtained from a parent or guardian on behalf of any participants under the age of 16. Xiangya Hospital is a large-scale, comprehensive, tertiary hospital with 3500 beds and nearly 3 million outpatients and emergency patients each year. The patients at this hospital mainly come from the central and southern regions of China. From 2011 to 2016, TTI serum markers were tested in 442,121 patients who planned to receive blood transfusions, surgeries or interventional procedures at Xiangya Hospital.Serum samples were assessed for antibodies to HIV types 1 and 2 (anti-HIV1/2), HBsAg, anti-HCV and anti-TP by chemiluminescence microparticle immunoassays (CMIA) on the ARCHITECT i2000SR . All the reagents were provided by Abbott Company and were approved by the State Food and Drug Administration of China. The system provided standardized quantitative results for HBsAg, expressed in international units (IU/ml), and values exceeding 0.05 IU/ml were considered positive for HBsAg. The presence or absence of anti-HIV 1/2, anti-HCV or anti-TP in the sample was determined by comparing the chemiluminescent signal in the reaction to the cut-off signal determined from an active ARCHITECT anti-HIV1/2, anti-HCV or anti-TP calibration curve, respectively. All the results of anti-HIV1/2, anti-HCV or anti-TP are expressed as the S/CO ratio, with S/CO < 1.0 indicating a nonreactive result and S/CO ≥ 1.0 indicating a reactive result. The samples positive for anti-TP were confirmed with identical test kits. A result was considered positive only when both results were positive. Serum from patients who screened positive for anti-HIV1/2 was collected again on the second day and re-examined by CMIA and colloidal gold immunochromatographic assay . The HIV antigen was fixed on the membrane with the nitrocellulose membrane as the carrier, and the samples were migrated along the solid-phase carrier. The quality-control band of the effective test had to be developed. The positive results and the quality-control band each showed a colored stripe on the membrane antigen site. As long as one method of re-examination result was positive, the serum samples were retested to confirm the existence of HIV infection by Western blot, which was performed in the laboratory of Hunan Provincial Centers for Disease Control and Prevention (CDC) . The opep-value < 0.05 was considered statistically significant.Statistical analyses were conducted using SPSS 19.0 statistics software. The chi-square test was applied to assess associations between categorical variants. The chi-square test for trends was used to analyze the differences in the seroprevalence of HBsAg, anti-HCV, anti-HIV and anti-TP among patients. A A total of 442,121 patients were screened for serum markers of the four TTIs during the study period. Of them, 218,378 (49.39%) were male, and 223,743 (50.61%) were female. The age of the study subjects ranged from 0 to 99 years (the average age was 43.85), and patients aged from 21 to 50 years accounted for 52.91% of all the subjects. The details are shown in Table The overall positivity of TTI serum markers was 15.35% (67875) and the positive rates of HBsAg, anti-HCV, anti-HIV and anti-TP were 10.98, 1.43, 0.16 and 2.78%, respectively (Table p < 0.001). HIV, HBV and HCV infection were more prevalent in males (p < 0.001). TP infection was more prevalent in females (p = 0.003) , especially in those from 41 to 50 years old (14.30%). The prevalence of HBsAg was lowest in those under the age of 20 years (2.34%). Most infections of TP were identified in aged patients. The positive rates of anti-TP significantly increased with increasing age and reached a peak (4.84%) in the age group older than 71 years. Likewise, the prevalence of HCV infection was high within those groups whose age exceeded 41 years. Overall, infection rates of HIV were relatively low, and those aged from 21 to 60 years had the highest positive rate of 0.19% (Table p < 0.001), while the positive rates of anti-HIV and anti-TP increased significantly (from 0.12% in 2011 to 0.19% in 2016 (p = 0.005) and from 2.32% in 2011 to 3.06% in 2016 (p < 0.001). The positive rate of anti-HCV remained relatively steady over the 6-year study period (p = 0.702).The positive rate of HBsAg decreased from 12.61% in 2011 to 10.27% in 2016 (p = 0.001), while in female patients, it remained relatively steady (p = 0.061)  Table .P = 0.032)  Fig. .Fig. 1Trp < 0.001), while the seroprevalence of HIV-HBV showed a slight increase from 0.03 to 0.05% (p = 0.020). The prevalence of triple or quadruple infection was rare and remained relatively steady.Of all the patients screened, 2788 0.63%) had multiple infections  and former plasma donors from 1985 to 2006 . HoweverThe Blood Donation Law in China prohibited the use of paid blood donation and required testing for the four TTIs discussed in this study in donors and in blood products for input. As a result, volunteer donors without payment now constitute the majority of blood donors. This transformation in the composition of blood donors has been associated with a gradual decrease in the prevalence of transfusion-transmitted HCV and HIV , 22. In The prevalence of HBV, HCV, HIV and TP infections in patients before blood transfusion in central and south China is relatively high. The overall infection rate of HBV is high but declining in young patients, while the infection rate of HIV is relatively low but showing an upward trend. Identification of these TTI serum markers in patients before blood transfusion is strongly recommended because it could facilitate the discovery of potential infectious diseases, reduce the chance of nosocomial infection of these diseases during medical treatment and provide useful epidemiological information about TTIs in certain areas.Additional file 1:Table S1. The distribution of TPPA positive specimens in different S/CO values in 2825 cases. (DOCX 30 kb)Preliminary screening and validation of TP antibodies in 2016.


Cryptosporidium parvum (studied here) and Cryptosporidium hominis are important causes of diarrhea in infants and immunosuppressed persons. C. parvum vaccine candidates, which are on the surface of sporozoites, include glycoproteins with Ser- and Thr-rich domains  and a low complexity, acidic protein (Cp23). Here we used mass spectrometry to determine that O-linked GalNAc is present in dense arrays on a glycopeptide with consecutive Ser derived from Gp40 and on glycopeptides with consecutive Thr derived from Gp20, a novel C. parvum glycoprotein with a formula weight of ~20 kDa. In contrast, the occupied Ser or Thr residues in glycopeptides from Gp15 and Gp900 are isolated from one another. Gly at the N-terminus of Cp23 is N-myristoylated, while Cys, the second amino acid, is S-palmitoylated. In summary, C. parvum O-GalNAc transferases, which are homologs of host enzymes, densely modify arrays of Ser or Thr, as well as isolated Ser and Thr residues on C. parvum vaccine candidates. The N-terminus of an immunodominant antigen has lipid modifications similar to those of host cells and other apicomplexan parasites. Mass spectrometric demonstration here of glycopeptides with O-glycans complements previous identification C. parvum O-GalNAc transferases, lectin binding to vaccine candidates, and human and mouse antibodies binding to glycopeptides. The significance of these post-translational modifications is discussed with regards to the function of these proteins and the design of serological tests and vaccines. C. parvum infects humans and cows, while C. hominis only infects humans .–21.18–21 to 324, residues . The ver to 45 V . The 45-, b6—b7) .O-glycan sites is occupied with a single HexNAc residue. We were unable to localize site occupancy in glycopeptides with 15–19 HexNAc residues, due to the labile nature of the O-glycans. Quite likely, the peptides modified with 15 to 19 HexNAc residues are a mixture of components having different occupancies.Because we saw little evidence for the presence of HexNAc-HexNAc, we assume that each of the 20 potential O-glycans from C. parvum sporulated oocyst proteins by reductive β–elimination, followed by GC/MS monosaccharide analysis versus sugar standards, showed that the HexNAc residues in the Gp40 glycopeptide and in glycopeptides of the other vaccine candidates are likely GalNAc ETSEAAATVDLFAFTLDGGK(240) with the addition of three HexNAc residues (m/z 204.0868) and full series of b and y ions, some of which retained a single HexNAc modification (marked with an asterisk). The product ion series y6* to y12* indicates Thr-235 is modified, and the series y13* to y15* suggests that either Thr-228 or Thr-235 is modified. The b3* ion indicates that either Thr-222 or Ser-223 is modified. Thus there is evidence for distribution of the three HexNAc residues over the four available sites in this peptide. In the glycopeptide with four HexNAc modifications, all possible O-glycan sites are occupied. Analyses of the fragmentation patterns of numerous other peptides (A non-tryptic glycopeptide of Gp15 AA-221 to 240), which results from cleavage of the Gp40/Gp15 precursor by the furin-like protease, contained one to four HexNAc modifications  18–21].–21.18–21 to 240, residues . Fragmenpeptides , both tr(87)EGEETDENTDETTTTTTTASPKPK(110) has 10 potential O-glycan sites and was found to be decorated with six to eight HexNAc residues  and numerous b and y ions retaining zero to two HexNAc residues (marked with asterisks). Because the vast majority of HexNAc residues were lost prior to peptide fragmentation, it was not possible to define the seven occupied sites or to determine whether the occupancy was heterogeneous. A second Gp20 glycopeptide (135)SSTTTTTTTAPVSSEDNKPEDSEDEK(160) with 12 potential O-glycan sites has a monoisotopic mass equal to that of the peptide with the addition of eight HexNAc residues , is a small, acidic, secreted protein with four domains with consecutive Thr residues, two of which are described here . The firresidues . For exaresidues . The 30-residues . Again gC. parvum vaccine candidates  KPTTTTTTTTTTTTK(623) with the addition of only three HexNAc residues, despite the presence of 12 available sites (m/z 204.0868) and numerous b and y ions containing zero to two HexNAc residues (marked with asterisks). Here again, because of the lability of the glycans, it was not possible to precisely define the occupied sites or to determine whether the occupancy was heterogeneous.Gp900 (cgd7_4020), which has an N-terminal signal peptide and a transmembrane domain near its C-terminus, is by far the largest of the peptide)  16, 17], 17C. paxception . The prele sites . The 30-m/z 895.4646 [M + 4H]4+ has a monoisotopic mass corresponding to the value calculated for the peptide (1712)NIVTEAAYGLPVDPK(1726) plus a single HexNAc residue GCSSSKPETK(11) similar to those modified by fatty acyl chains in the host and other apicomplexans  and palmitate (m/z 239.2370), as well as the charge reduced [M + H]1+ molecular ion with loss of palmitate (m/z 1233.6782) or myristate (m/z 1261.7001). The presence of a complete y -ion series and a partial b -ion series allowed us to assign myristate to the N-terminal Gly and palmitate to the Cys. We believe the example given is what is present on the native protein. The peptides where palmitate is absent and Cys is carbamidomethylated or palmitate modifies Ser residues arose during sample processing . F. F(2)GCSalmitate . FragmenO-linked HexNAc  is a widespread modification of C. parvum vaccine candidates  (TIFF)Click here for additional data file.S2 FigC. parvum oocyst glycoproteins are compared with results observed for the GlcNAc and GalNAc standards, which were similarly treated. Extracted ion chromatograms of m/z 101 are shown on the left. Electron impact mass spectra of the standards and the product from reductive β-elimination of C. parvum oocyst glycoproteins are shown on the right. The sugar released from C. parvum is assigned as GalNAc, because the retention time (34 min) and EI mass spectrum both match those of the standard GalNAc. The EI mass spectra of the components eluting at positions marked with one asterisk (*) and two asterisks (**) do not correspond to sugar derivatives.GC/MS data obtained for the deuteroreduced and permethylated glycan released by reductive β-elimination of (TIFF)Click here for additional data file.S3 Fig3+m/z 732.0284 has a monoisotopic mass corresponding to the peptide (609)KPTTTTTTTTTTTTK(623) with the addition of three HexNAc residues (Δ -0.1 ppm). There is a prominent HexNAc oxonium ion (m/z 204.0868) and a full series of b and y ions, some of which contain one (*) or two (**) HexNAc residues. The loss of HexNAc residues made it impossible to localize occupied sites. Charge-reduced aglycon peptide ions are observed in the spectrum: ǂ = [M + 2H]2+m/z 792.9193 and † = [M + H]1+m/z 1584.8348.The precursor ion [M + 3H](TIFF)Click here for additional data file.S4 FigThis figure presents an example of an MS/MS spectrum initially assigned by PEAKS DB, then manually verified and re-annotated using the in-house software GlycReSoft. GlycReSoft is capable of discovery and annotation, but only the annotation capabilities were utilized here.(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.